Bacteriophage preparations and methods of use thereof

ABSTRACT

Disclosed herein are purified bacteriophage preparations that effectively lyse a plurality of  Clostridium  species strains, in particular  C. perfringens, C. septicum  and  C. difficile . In one embodiment, a purified bacteriophage preparation includes four or more  C. perfringens -specific bacteriophage, wherein each bacteriophage has lytic activity against at least five  Clostridium  species strains. In another embodiment, the purified bacteriophage preparation includes five or more  C. perfringens -specific bacteriophage. The invention also relates to the use of purified bacteriophage preparation in combination with antibiotics for the treatment of animals including poultry. The invention also relates to the use of the purified bacteriophage preparations as treatments effective against antibiotic-resistant strains of  Clostridium.

CROSS REFERENCE OF RELATED PATENT APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/334,863 filed on Dec. 15, 2008 which claims priority to U.S.Provisional Application Ser. No. 61/013,325 filed on Dec. 13, 2007, bothof which are incorporated in their entirety by reference herein.

BACKGROUND

Antibiotic use enhances the growth of healthy domesticated poultry andlivestock. Although extensive bans and restrictions have not beenimplemented in the United States as they have in the E.U. and othercountries, pressure for antibiotics alternatives has increased due toconcerns of increasing antibiotic resistance among food borne bacteria.Banning or markedly reducing the agricultural and farm-veterinary use ofantibiotics may have a profound negative impact on the safety of foodsand on the treatment of sick flocks or herds of domesticated livestock,however. Thus, effective, safe and environmentally friendlyalternative(s) to antibiotics are needed to address these concerns andneeds.

Viruses that kill bacteria were first identified in the early part ofthe 20^(th) century by Frederick Twort and Felix d'Herelle who calledthem bacteriophages or bacteria-eaters (from the Greek phago meaning toeat or devour). Because of their remarkable antibacterial activity,phages were used to treat disease of economically importantanimals/domesticated livestock almost immediately after their discovery,and therapeutic applications for humans closely followed. However, withthe advent of antibiotics, phage therapy gradually fell out-of-favor inthe United States and Western Europe, and virtually no subsequentresearch was done on the potential therapeutic application of phages forbacterial diseases of humans or animals. The emergence ofantibiotic-resistance in bacteria, however, has rekindled interest intherapeutic bacteriophages. Phage therapy may have a positive impact onhuman health by improving the safety of foods in the U.S.A. andelsewhere, and by helping to reduce safely the use of antibiotics inagribusiness.

Among the bacteria that cause significant morbidity and mortality inchickens, C. perfringens is one of the most notorious pathogens. Inchicken C. perfringens infections are often manifested as necroticenteritis that occur later in the production cycle, often following acoccidial infection or other insult to the gastrointestinal tract. It isthus desirable to develop bacteriophage preparations suitable to reducemorbidity and mortality in chickens.

C. septicum is an anaerobic bacterium which causes or contributes to gasgangrene and malignant edema in animals and people, usually followingdirect contact of a traumatic wound. C. septicum multiplies locally anddisseminates throughout the animal's body, producing local lesions andsigns of toxemia (Songer, 1996). Specifically for turkeys and chickens,C. septicum is implicated in gangrenous dermatitis (or cellulitis), aneconomically important disease (Tellez et al., 2009; NTF, 2007).Typically, antibiotics are used to treat or prevent C. septicuminfections in poultry (Songer, 1996, NTF, 2007).

Clostridium difficile is well established as a pathogen of horses,calves, and pigs, as well as poultry. Clostridium difficile is a normalinhabitant of the gastrointestinal tract of many species of mammals andhas been isolated from bird feces; it is the most common pathogenicenteric clostridial organism in humans.

SUMMARY

Disclosed herein are purified bacteriophage preparations thateffectively lyse a plurality of strains of a Clostridium species. In oneembodiment, a purified bacteriophage preparation comprises four or moreC. perfringens-specific bacteriophage, wherein each bacteriophage haslytic activity against at least five strains of a Clostridium species.In another embodiment, the purified bacteriophage comprises five or moreC. perfringens-specific bacteriophage. The bacteriophage preparationsare effective against both antibiotic susceptible andantibiotic-resistant strains of Clostridium including C. perfringens, C.septicum and C. difficile.

Disclosed herein are purified bacteriophage preparations thateffectively lyse a plurality of C. perfringens strains. In oneembodiment, a purified bacteriophage preparation comprises four or moreC. perfringens-specific bacteriophage, wherein each bacteriophage haslytic activity against at least five C. perfringens strains. In anotherembodiment, the purified bacteriophage comprises five or more C.perfringens-specific bacteriophage.

Disclosed herein are purified bacteriophage preparations thateffectively lyse a plurality of C. septicum strains. In one embodiment,a purified bacteriophage preparation comprises four or more C.perfringens-specific bacteriophage, wherein each bacteriophage has lyticactivity against at least five C. septicum strains. In anotherembodiment, the purified bacteriophage comprises five or more C.perfringens-specific bacteriophage.

Disclosed herein are purified bacteriophage preparations thateffectively lyse a plurality of C. difficile strains. In one embodiment,a purified bacteriophage preparation comprises four or more C.perfringens-specific bacteriophage, wherein each bacteriophage has lyticactivity against at least five C. septicum strains. In anotherembodiment, the purified bacteriophage comprises five or more C.perfringens-specific bacteriophage.

In another embodiment, a method of reducing chicken mortality due to C.perfringens infections comprises administering a purified bacteriophagepreparation four or more C. perfringens-specific bacteriophage, whereineach bacteriophage has lytic activity against at least five strains of aClostridium species including but not limited to C. perfringens, C.septicum or C. difficile.

In another embodiment, a method of selecting a C. perfringens hoststrain suitable to propagate bacteriophage from a plurality of teststrains comprises microbiologically confirming one test strain from theplurality of test strains as a C. perfringens species to produce aconfirmed strain; associating the confirmed strain with a poultrydisease to produce a disease-associated strain; and applying one or moreadditional selective criterion to the disease-associated strain selectedfrom minimal antibiotic resistance and absence of animal-virulencemarkers other than for C. perfringens to produce the C. perfringens hoststrain suitable to propagate bacteriophage.

In yet another embodiment, a method of producing a bacteriophagecocktail comprises mixing four or more C. perfringens-specificbacteriophage, wherein each bacteriophage has lytic activity against atleast five strains of a Clostridium species including but not limited toC. perfringens, C. septicum or C. difficile.

The present invention also relates to the use of bacteriophagepreparations in combination with antibiotics. The bacteriophagepreparations when used in combination with antibiotics againstClostridium species preferably C. perfringens, C. septicum, or C.difficile which cause necrotic enteritis, gangrenous dermatitis,cellulitis, or enteric diarrheal diseases provide complementary(additive and/or synergistic) lytic activity. The bacteriophagepreparations of the present invention can be combined with antibioticpreparations to make a single composition for administration to animalsfor growth enhancement and/or therapeutic purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a phylogenetic analysis of C. perfringens strains.

FIG. 2 is a dendrogram portraying genetic diversity of various C.perfringens strains based on SmaI-digested PGFE patterns of C.perfringens DNA.

FIG. 3 shows phage plaques produced by representative bacteriophageinfecting C. perfringens strain Cp 42.

FIG. 4: Pulsed-field gel electrophoresis analysis of undigested phageDNA isolated from each monophage. Lane M, Low Range PFG Marker (NEB);lane 1, CPAS-7; lane 2 CPAS-15, lane 3, CPAS-16; lane 4, CPLV-42; lane 5CPTA-12; lane 6, CPAS-37.

FIG. 5 shows XmnI digested phage DNA from each monophage. Lane M,GeneRuler DNA Ladder Mix (Fermentas); lane 1, CPLV-42; lane 2, CPAS-7,lane 3, CPAS-15; lane 4, CPTA-37; lane 5 CPAS-12; lane 6, CPAS-16.

FIG. 6 shows structural protein profiles for each C. perfringensmonophage. Lane 1, CPLV-42; lane 2, CPAS-12, lane 3, CPAS-7; lane 4,CPAS-16; lane 5 CPAS-15; lane 6, CPTA-37.

FIG. 7 shows electron micrographs of C. perfringens bacteriophages.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Disclosed herein are purified bacteriophage preparations thateffectively lyse a plurality of Clostridium species strains including C.perfringens, C. septicum or C. difficile. Lysis of particular strains isdemonstrated by the drop-on-lawn method, which is standard in the art.The bacteriophage preparations are suitable to reduce morbidity andmortality in chickens.

In one embodiment, a purified bacteriophage preparation comprises fouror more C. perfringens-specific bacteriophage, wherein eachbacteriophage has lytic activity against at least five C. perfringensstrains. In another embodiment, the purified bacteriophage comprisesfive or more C. perfringens-specific bacteriophage.

In one embodiment, a purified bacteriophage preparation comprises fouror more C. perfringens-specific bacteriophage, wherein eachbacteriophage has lytic activity against at least five C. septicumstrains. In another embodiment, the purified bacteriophage comprisesfive or more C. perfringens-specific bacteriophage.

In one embodiment, the bacteriophage preparation comprises CPAS-12(accession number PTA-8479), CPAS-15 (accession number PTA-8480),CPAS-16 (accession number PTA-8481) and CPLV-42 (accession numberPTA-8483). In another embodiment, the bacteriophage preparation consistsessentially of CPAS-12, CPAS-15, CPAS-16 and CPLV-42. In yet anotherembodiment, the bacteriophage preparation consists of CPAS-12, CPAS-15,CPAS-16 and CPLV-42.

In another specific embodiment, the bacteriophage preparation comprisesCPAS-7 (accession number PTA-8478), CPAS-12, CPAS-15, CPAS-16 andCPLV-42 (accession number PTA-8483). In another embodiment, thebacteriophage preparation consists essentially of CPAS-7, CPAS-12,CPAS-15, CPAS-16 and CPLV-42. In yet another embodiment, thebacteriophage preparation consists of CPAS-7, CPAS-12, CPAS-15, CPAS-16and CPLV-42.

In one embodiment, the C. perfringens strains are ATCC strain 25768,ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124,ATCC strain PTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRLstrain B-50145, and combinations comprising one or more of the foregoingstrains. In a specific embodiment, the at least five C. perfringensstrains comprise ATCC strain 3624 and ATCC strain 9856.

In one embodiment, the bacteriophage preparations are characterized bytheir specificity and effectiveness against C. perfringens strains. Inone embodiment the purified bacteriophage preparation lyses greater thanor equal to 85% of at least 40 screened C. perfringens strains, whereinthe bacteriophage preparation is incapable of infecting at least 10strains of E. coli, L. monocytogenes, S. enteric and P. aeruginosa. Inanother embodiment the purified bacteriophage preparation lyses greaterthan or equal to 85% of at least 45 screened C. perfringens strains. Inyet another embodiment, each of the individual C. perfringens-specificbacteriophage lyses 15% to 90% of the screened C. perfringens strains.

In one embodiment, the bacteriophage preparations are characterized bytheir specificity and effectiveness against C. septicum strains. In oneembodiment the purified bacteriophage preparation fully or partiallylyses greater than or equal to 85% of at least 7 screened C. septicumstrains, wherein the bacteriophage preparation is incapable of infectingat least 10 strains of E. coli, L. monocytogenes, S. enteric and P.aeruginosa. In another embodiment the purified bacteriophage preparationfully lyses greater than or equal to 25% of at least 7 screened C.septicum strains. In yet another embodiment, each of the individual C.perfringens-specific bacteriophage lyses 15% to 90% of the screened C.septicum strains.

The present invention also relates to the use of bacteriophagepreparations in combination with antibiotics. The bacteriophagepreparations when used in combination with antibiotics againstClostridium species preferably C. perfringens, C. septicum, or C.difficile which cause necrotic enteritis, gangrenous dermatitis,cellulitis, or enteric diarrheal diseases provide complementary(additive and/or synergistic) lytic activity. In one embodiment thebacteriophage preparations could be applied to animals also beingadministered antibiotics for growth enhancement and/or therapeuticpurposes. In another embodiment the bacteriophage preparations can becombined with the antibiotics in a single preparation provided toanimals for growth enhancement and/or therapeutic purposes.

The bacteriophage preparations are effective against both antibioticsusceptible and antibiotic-resistant strains of Clostridium including C.perfringens, C. septicum and C. difficile. Agricultural or medicalstrains having natural tolerance or acquired resistance to antibiotics,may have reduced therapeutic effectiveness if treated with the same orsimilar antibiotic classes to which they are tolerant, but would bepredicted to be more susceptible to the phage cocktail INT-401 if ananimal or person were alternatively or concurrently treated withINT-401. This pattern may allow a) enhanced killing or inhibition ofdisease causing Clostridium spp. in animals or humans if combined, b)rotating or strategically dosing different levels of treatment ofantibiotics and phage in order to efficiently reduce or eliminate thetotal burden of disease-causing Clostridium spp. in the animal orperson's gastrointestinal tract at different disease stages oranimal/human ages. Concurrent treatment of antibiotics plusbacteriophage could allow enhancement of killing or inhibitory action ofdisease-causing bacteria, as well as reducing the likelihood ofindependent phage or antibiotic resistance selection due to theirdifferent and complementary modes of action, in animals or people.

The bacteriophage preparations of the present invention, preferablyINT-401, can lyse greater than 25% of bacitracin resistant and/ormacrolide-resistant (tylosin-resistant) strains of C. perfringens. Thebacteriophage preparations of the present invention can lyse greaterthan 50% of bacitracin resistant and/or macrolide-resistant(tylosin-resistant) strains of C. perfringens. The bacteriophagepreparations of the present invention can lyse antibiotic-resistantbacteria to the same or greater degree as antibiotic-susceptible types.

The term “purified” in reference to a bacteriophage or bacteriophagepreparation does not require absolute purity (such as a homogeneouspreparation). Instead, it is an indication that the bacteriophage orbacteriophage preparation is relatively more pure than in the naturalenvironment (compared to the natural level, this level should be atleast 2-5 fold greater, e.g., in terms of mg/mL). Purification isaccording to any method known to those of ordinary skill in the art thatwill result in a preparation of bacteriophage substantially free fromother nucleic acids, proteins, carbohydrates, lipids, or subcellularorganelles. Individual bacteriophage may be purified to electrophoretichomogeneity. Purification of at least one order of magnitude, preferablytwo or three orders, and more preferably four or five orders ofmagnitude is expressly contemplated.

Purity of phage stocks can be determined by pulsed-field gelelectrophoresis (PFGE) of uncut DNA. In one embodiment, approximately100-200 ng of the phage DNA is electrophoresed in a 1% SeaKem GoldAgarose (Cambrex, Rockland, Me.) gel with 0.5× Sodium boric acid (1×SB:10 mM sodium hydroxide pH adjusted to 8.5 with boric acid) buffer at 14°C. in a CHEF Mapper XA PFGE apparatus (Bio-Rad Laboratories, Hercules,Calif.). The run time is 12 hours with a voltage of 6 V/cm and alinearly ramped pulse time of 0.06 s to 8.53 seconds. The gels werestained with ethidium bromide and visualized with UV light.

In one embodiment, endotoxin levels for lots of phage produced aredetermined by a Limulus amoebocyte lysate (LAL) assay using achromogenic assay (QCL-1000 Kit, BioWhittaker, Walkersville, Md.)following manufacturers' recommendations. A standard curve with E. coliendotoxin (supplied with kit) ranging from 0.1 to 1.0 endotoxin units(EU) per milliliter endotoxin is constructed for each assay by plottingthe optical density at 405 nm (OD₄₀₅) versus EU/ml. Phage samples areassayed diluted in sterile water (USP, Baxter, Deerfield, Ill.) and theabsorbance at 405 nm was measured with a μQuant (Bio-Tek Instruments,Inc., Winooski, Vt.) microplate reader. Endotoxin concentrations foreach sample are calculated by linear regression from the standard curve.Standards and samples are analyzed in triplicate.

In one embodiment, carbohydrate content for all lots of phage producedis determine by an anthrone method. A standard curve with glucoseranging from 10 to 250 μg/ml is constructed for each assay by plottingoptical density at 625 nm (OD₆₂₅) versus μg/ml glucose concentration.Phage samples are assayed in PBS and the absorbance at 625 nm wasmeasured with a μQuant (Bio-Tek Instruments, Inc., Winooski, Vt.)microplate reader. Carbohydrate concentrations for each sample arecalculated by linear regression from the standard curve. Standards andsamples are analyzed in triplicate.

In one embodiment, the total protein content for all lots of phageproduced is determine by a bicinchoninic acid (BCA) assay using acolorometric assay (BCA™ Protein Assay kit, Pierce, Rockford, Ill.)following manufacturers' recommendations. A standard curve with bovineserum albumin ranging from 20 to 2000 μg/ml protein is constructed foreach assay by plotting optical density at 562 nm (OD₅₆₂) versus μg/mlBSA. The absorbance at 562 nm was measured with a μQuant (Bio-TekInstruments) microplate reader. Total protein concentrations for eachsample are calculated by linear regression from the standard curve.Standards and samples are analyzed in triplicate.

The term “strain” means bacteria or bacteriophage having a particulargenetic content. The genetic content includes genomic content as well asrecombinant vectors. Thus, for example, two otherwise identicalbacterial cells would represent different strains if each contained avector, e.g., a plasmid, with different phage open reading frames.

By “treatment” or “treating” is meant the administering a bacteriophagepreparation for prophylactic and/or therapeutic purposes. The term“prophylactic treatment” refers to treating an animal that is not yetinfected but is susceptible or otherwise at risk of a bacterialinfection. The term “therapeutic treatment” refers to administeringtreatment to an animal already suffering from infection.

The term “bacterial infection” means an invasion of the host organism,animal or plant, by pathogenic bacteria. This includes excessive growthof bacteria which are normally present in or on the body of an organism,but more generally, a bacterial infection is any situation in which thepresence of a bacterial population(s) is damaging to a host organism.Thus, for example, an organism suffers from a bacterial infection whenexcessive numbers of a bacterial population are present in or on theorganism's body, or when the effects of the presence of a bacterialpopulation(s) is damaging to the cells, tissue, or organs of anorganism.

The terms “administer”, “administering”, and “administration” refer to amethod of giving a dosage of a bacteriophage preparation to an organism.Suitable methods include topical, oral, intravenous, transdermal,intraperitoneal, intramuscular, or intrathecal. The preferred method ofadministration varies depending on various factors, e.g., the componentsof the bacteriophage preparation, the site of potential or actualbacterial infection, the bacterium involved, and the infection severity.

In the context of treating a bacterial infection a “therapeuticallyeffective amount” or pharmaceutically effective amount” indicates anamount of a bacteriophage preparation which has a therapeutic effect.This generally refers to the inhibition, to some extent, of the normalcellular functioning of bacterial cells that render or contribute tobacterial infection.

The doses of bacteriophage preparation that is useful as a treatment isa “therapeutically effective amount”. Thus, as used herein, atherapeutically effective amount means an amount of a bacteriophagepreparation that produces the desired therapeutic effect as judged byclinical trial results. This amount can be routinely determined by oneskilled in the art and will vary depending on several factors, such asthe particular bacterial strain involved and the particularbacteriophage preparation used.

In one embodiment, a bacteriophage preparation optionally includes oneor more pharmaceutically acceptable excipients. In one embodiment, theexcipient is a water-conditioning agent, for example agent suitable forwater dechlorination and/or phage stabilization. Such agents areinnocuous to the bacteriophage cocktail, but when added prior to orsimultaneously with C. perfringens bacteriophage or bacteriophagecocktails, act to dechlorinate municipal levels of chlorine, which ifuntreated would kill or significantly reduce the viability of thebacteriophage or bacteriophage cocktail. Exemplary water-conditioningagents include amino acids and/or salts which help to normalize the pHand ionic balance of the bacteriophage cocktail, when added to diversewater sources used for the animal's drinking and for phage delivery. Inone embodiment, the water-conditioning agent is a 50 mMcitrate-phosphate-thiosulfate (CPT) buffer, comprising 40 mg sodiumthiosulfate, 6.0 gm disodium phosphate (anhydrous), 1.1 gm citric acid(anhydrous) per liter deionized water pH 7.0. By includingwater-conditioning agents in the cocktail or adding separately to thetreatment water, the water-conditioning agents act to both stabilize andprotect the bacteriophage cocktail in a commercial preparation suitablefor routine field use.

A method of producing a bacteriophage cocktail comprises mixing four ormore C. perfringens-specific bacteriophage, wherein each bacteriophagehas lytic activity against at least five C. perfringens strains. Inanother embodiment, a method of producing a bacteriophage cocktailcomprises mixing five or more C. perfringens-specific bacteriophagewherein each bacteriophage has lytic activity against at least five C.perfringens strains.

Once C. perfringens cocktail have been selected, further testing can beemployed to refine the specificity of the cocktail. In one embodiment, abacteriophage cocktail is tested against additional C. perfringensstrains which have antibiotic resistance genes to test the phagecocktail against antibiotic-resistant strains and evaluate thecocktail's potential use in the field against antibiotic-resistantClostridia. In another embodiment, a bacteriophage cocktail is testedagainst C. perfringens strains derived from other animal species otherthan chickens to further evaluate and define the host range of thestrain or strains to include additional animal species (e.g., swine,cattle, turkey, sheep, exotics, dogs, cats and the like). In anotherembodiment, a bacteriophage cocktail is tested against Clostridium ofdifferent species (i.e., other than C. perfringens such as but notlimited to C. perfringens, C. septicum and C. difficile. In yet anotherembodiment, a bacteriophage cocktail is tested against additionalgram-positive bacteria (e.g., both reference strains andanimal-associated types, aerobic and anaerobic) to further evaluate anddefine the host range. In another embodiment, a bacteriophage cocktailis tested against additional gram-negative and gram-variable bacteria tofurther evaluate host range. In another embodiment, a bacteriophagecocktail is tested for pre-conditioning or additive formulations, usingdifferent levels of stabilizing and dechlorinating water-conditioningagents, for optimally maintaining the viability of the phage cocktailunder a wide range of water types and chlorination levels as may beexpected in field usage conditions.

In another embodiment, the method further comprises testing thepotential of the bacteriophage cocktail for the development of intrinsicphage resistance in the target host. Testing includes challenging testC. perfringens, C. septicum and/or C. difficile as well as otherClostridium species host strains with individual and/or combinedbacteriophage cocktail phage over several cycles, and ascertaining therate of resistance development toward the individual phages as well asthat of the combination(s) of phages. It is anticipated that thecombination bacteriophage cocktail will have significantly lessdevelopment of resistance against a given individual host strain. Anoptimum combination of bacteriophages may be further elucidated usingknown mathematical optimization techniques or software packages(Box-Hunter, Latin Squares, Taguchi, Simplex, etc.) as applied to thebacteriophage resistance data generated from such experimentation.

Advantages of bacteriophage therapy include high bactericidal activity,high selectivity permitting targeting of specific pathogens whileleaving desirable bacterial flora intact, specifically for prokaryoticcells, and environmental benignity. In livestock and poultryapplications, bacteriophage have the advantage of specificity thatshould not select for phage-resistance in non-targeted bacterialspecies, the possible emergence of resistance against phages will notaffect the susceptibility of the bacteria to antibiotics used to treathumans, and unlike antibiotics, phage preparations can readily bemodified in response to changes in bacterial pathogen populations orsusceptibility.

The poultry and livestock industries use antibiotics for three mainpurposes: (i) prophylactic ally, to prevent disease in flocks, herds,etc., (ii) to treat sick livestock, and (iii) to improve digestion andutilization of feed, which often results in improved weight gain.Antibiotics used in the latter setting are often referred to as“growth-promoting antibiotics” or GPAs. Most GPAs are not commonly usedin human medicine, and they are usually administered, in small amounts,to poultry and other livestock via food. Bacteriophages can effectivelyreplace and/or reduce the use of antibiotics in all three of theabove-mentioned settings.

Among the bacteria that cause significant morbidity and mortality inchickens, C. perfringens is one of the most notorious pathogens. Inorder to identify effective bacteriophage for a generically diversepopulation of C. perfringens strains, isolates of C. perfringens werefirst identified. In order to identify effective bacteriophage, it isuseful to identify C. perfringens strains that affect poultry at variouslocations within the United States. As shown herein, forty-one strainsof C. perfringens were isolated from various sources and characterizedby pulsed-filed gel electrophoresis (PFGE) typing. (FIG. 1) Among the 35strains subjected to PFGE, phylogenetic analysis showed that thesestrains clustered into 15 heterogenic groups. (FIG. 2) Among these 15PFGE types, P6 is the predominant type (10 strains) followed by P4 (6strains). Additional C. perfringens strains may be obtained frompublicly available collections.

One important factor in the identification of bacteriophage is theselection of C. perfringens strains suitable for their identification.In one embodiment, a method of selecting a C. perfringens host strainsuitable to propagate bacteriophage from a plurality of test strains asa C. perfringens species to produce a confirmed strain; associating theconfirmed strain with a poultry disease to produce a disease-associatedstrain; applying one or more additional selective criterion to thedisease-associated strain selected from minimal antibiotic resistanceand absence of animal-virulence markers other than for C. perfringens toproduce the C. perfringens host strain suitable to propagatebacteriophage. In one embodiment, the selection criterion is minimalantibiotic resistance and the antibiotic resistance is tetracycline,ampicillin, tylosin, erythromycin, lincomycin, chloramphenicol or otherdrug resistance. The selection of strains absent from antibioticresistance minimizes the potential transduction of plasmid orchromosomal-borne antibiotic resistance genes, into the subsequentbacteriophage cocktail. The advantage of this applied criterion, is toin advance, limit any potential resistance genes in a bacteriophagecocktail preparation. The selective criterion used for these phagecocktail host strains, are a unique extension of a unique library of C.perfringens strains, combined with microbiological knowledge ofantibiotic resistance, along with the skills in running antibioticsusceptibility tests to ascertain the resistance profiles of thesubmitted host strain.

Six novel bacteriophages of the Siphoviridae or Miroviridae familiesthat infect Clostridium perfringens were isolated from environmentalwater or sewage sources. Phage were characterized, for example, at boththe protein and nucleic acid level. The optimal host strain forpropagation of each bacteriophage is identified and all phage arepreferably negative for endogenous phage. In addition, eachbacteriophage is characterized by PFGE, RAPD, SDS-PAGE, and otherapproaches. Stocks of all six monophages and their respective hoststrains are made for use in characterization and production of eachphage.

The C. perfringens-specific monophages are capable of specificallyinfecting C. perfringens strains and are not capable ofinfecting/growing on E. coli, L. monocytogenes, S. enteric and P.aeruginosa. As used herein, the term C. perfringens-specific refers tobacteriophage and bacteriophage preparations that are capable ofinfecting a plurality of C. perfringens strains and may be capable ofinfecting other Clostridium species but are incapable of infecting atleast 10 strains of E. coli, L. monocytogenes, S. enteric and P.aeruginosa.

Six bacteriophages that infect Clostridium perfringens were sequenced.Five of the six phages are sequenced, and each predicted open readingframe is identified in each genome. Each of the predicted genes wasannotated. None of the 17 undesirable genes (Table 5.1.1) is found inthe genomes of any of the five phages for which sequences wereavailable.

Two phage cocktails, INT-401 (CPAS-7, CPAS-12, CPAS-15, CPAS-16 andCPLV-42) and INT-402 (CPAS-12, CPAS-15, CPAS-16 and CPLV-42), areprepared from five of the six monophages isolated. Both cocktails areeffective in killing greater than 85% of the 46 C. perfringens strainsscreened. INT-401 was selected for use in proof-of-principle efficacystudies designed to determine the prevention of necrotic enteritis in C.perfringens challenged broiler chickens.

Oral Gavage of Test Article (INT-401 phage cocktail) to bird on the dayof challenge (Day 14) and for the next four days significantly reducedmortality due to NE. Growth performance in this group was numericallyequivalent to the non-challenged control, and appeared to be bettercompared to the challenged, but phage-untreated chickens. Given the factthat many chickens are naturally colonized with C. perfringens, thelatter observation warrants further elucidation, to better examine thepossible growth performance-enhancing benefits of the phage preparation.

Two of the three “in ovo” treatments had numerically reduced NEmortality (9.6 and 14.8%) when compared with the Challenged control(25.9%).

Oral Gavage of Test Article prior to challenge, or spray of Test Articleto chicks at the hatchery, was ineffective in preventing NE mortalitydue to C. perfringens challenge.

The results of the studies herein suggest that C. perfringens-specificphage preparation can be effective in significantly reducing chickenmortality due to C. perfringens infections in chickens such as thosecausing necrotic enteritis when administered shortly after the bacterialchallenge. Further dosing- and delivery-optimization studies arewarranted, together with fine-tuning of the product for the optimalefficacy.

Exemplary means of administration of the bacteriophage preparations areoral administration, intramuscular injection, subcutaneous injection,intravenous injection, intraperitoneal injection, eye drop, nasal sprayand the like. When the subject to be treated is a bird, the bird may bea hatched bird, including a newly hatched (i.e., about the first threedays after hatch), adolescent, and adult birds. Birds may beadministered the vaccine in ovo, as described in U.S. Pat. No. 4,458,630to Sharma, for example, incorporated herein by reference.

In one embodiment, the bacteriophage preparation is administered in ananimal feed such as poultry feed. The bacteriophage preparation isprepared in a number of ways. For instance, it can be prepared by simplymissing the different appropriate compounds to produce the bacteriophagepreparation. The resulting bacteriophage preparation can then be withermixed directly with feed, or more conventionally impregnated onto acereal-based carrier material such as milled wheat, maize or soya flour.Such an impregnated carrier constitutes a feed additive, for example.

The bacteriophage preparation may be mixed directly with the animalfeed, or alternatively mixed with one or more other feed additives suchas vitamin feed additive, a mineral feed additive, or an amino acid feedadditive. The resulting feed additive including several different typesof components can then be mixed in an appropriate amount with the feed.It is also possible to include the bacteriophage preparation in theanimal's diet by incorporating it into a second (and different) feed ordrinking water which the animal also has access to. Accordingly, it isnot essential that the bacteriophage preparation is incorporated intothe usual cereal-based main feed of an animal.

In one embodiment, included are the methods of identifying an optimizedfield delivery modes and conditions for phage cocktail applications. Inone embodiment, an optimized administration condition is wateradministration, for up to 3 days at temperatures up to 50° C.

The bacteriophage preparation can be used for a wide variety of animals,but the use of the bacteriophage preparation is particularly preferredin domestic animals and farm livestock. Animals which may in particularbenefit from the bacteriophage preparation include poultry (such aschickens, turkeys, ducks and geese), ruminants (such as cattle, horseand sheep), swine (pigs), rodents (such as rabbits) and fish. Thebacteriophage preparation is particularly useful in broiler chickens.

EXAMPLES Example 1 Characterization of Clostridium perfringens Isolates

Media: Brain Heart Infusion (BHI) broth or BHI agar was used to grow allisolates. All media were obtained from EMD Chemicals, Gibbstown, N.J.

Microoganisms: Forty-two C. perfringens strains were employed. Onestrain (Cp20) did not grow and was excluded from further analysis. Aspart of the collection process, isolates were checked for purity andfrozen at −80° C. in 30% glycerol. Most of the work was performed in ananaerobic chamber (Plas-Labs, Lansing, Mich.), that contained a 90%N₂-5% H₂-5% CO₂ atmosphere.

Bacteriophage: All bacteriophage were isolated from environmental watersources.

Phage Isolation: Samples of water collected for the isolation of phagewere mixed with 10×BHI broth, inoculated with a single C. perfringensstrain of interest and incubated anaerobically at 37° C. overnight. Thesamples were centrifuged (8,000×g, 10 min) to remove the bacterial cellsand sterile filtered (0.22 μm Stericup™, Millipore, Bedford, Mass.).Filtrates were serially diluted in BHI broth and tittered usingsoft-agar overlay method. Briefly, dilutions of each filtrate were mixedwith log-phase bacterial culture, incubated 37° C. for 10 minutes,molten soft-agar added, poured onto BHI agar plates and incubatedanaerobically at 37° C. overnight. Individual plaques were picked fromthe overlay plated and tittered a second time as an initial step inensuring that each phage was pure.

Screening for Endogenous Phage: Clostridium perfringens strains used forpropagating the plaques were screened for endogenous bacteriophage bythe drop lawn method. Liquid cultures of host strains were grownovernight, centrifuged (9,500×g, 5 minutes) to remove bacteria andfiltered through a 0.22 μm syringe filter (Millipore). The same strainswere grown in BHI broth to an OD₆₀₀ of 0.1-0.3. Two hundred microlitersof each screening strain was mixed with molten soft-agar and poured ontoa BHI agar plate. After the soft-agar hardened 10 μl of each host strainfiltrate was spotted onto the plates with the screening strains. Lyticactivity was observed after overnight anaerobic incubation at 37° C.

Clostridium perfringens Host Strain Typing: The 41 C. perfringensstrains received were typed by PFGE using the National MolecularSubtyping Network (PulseNet) standard protocol. C. perfringens strainswere grown on BHI agar overnight anaerobically at 37° C. and suspendedin 75 nM NaCl-25 mM EDTA (pH 8.0) (CSB) buffer to an OD₆₀₀ of 1.3-1.4.The bacterial cells were embedded in 1.2% SeaKem® Gold Agarose (Cambrex,Rockland, Me.) by mixing equal volumes (0.4 mL) of the cell suspensionand melted agarose made in TE buffer. Plugs were made in 1.5-mm thickmolds (Bio-Rad Laboratories, Hercules, Calif.) and solidified at 4° C.the cells were lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 50 mMEDTA [pH 8.0], 1% laurylsarcosine, and proteinase K [1 mg/ml]) at 55° C.overnight. The plugs were washed at 54° C. with shaking three times for15 minutes each in sterile water and then three times in Te buffer. Theplugs were stored at 4° C. in Te buffer.

Plugs were equilibrated with restriction endonuclease buffer at 25° C.overnight. The plugs were digested with SmaI (New England Biolabs,Beverly, Mass.) according to manufacturers' recommendations overnight.Restriction fragments were separated by electrophoresis through 1%agarose gel in 0.5× Tris-borate-EDTA (10×TBE, EMD Chemicals) with 1 mMthio-urea at 14° C. in a CHEF Mapper XA PFGE apparatus (Bio-RadLaboratories) The run time was 20 hours with a voltage of 6 V/cm and alinearly ramped pulse time of 0.4 seconds to 40 seconds. The range sizeanalyzed was 40-1,400 kilobases.

Data Handling and Analysis: A large zone of clearing (lytic activity)produced lawns of any of the C. perfringens strains where culturefiltrate was applied was considered positive for endogenous phage.

PFGE Results: Forty-one strains of C. perfringens were isolated fromvarious sources and characterized by pulsed-field gel electrophoresis(PFGE) typing. (Table 1) Among the 35 strains (one strain did not growand six were not type-able due to nuclease problems) subjected to PFGE,phylogenetic analysis showed that these strains clustered into 15heterogenic groups. (FIG. 1) Among these 15 PFGE types, P6 is thepredominant type (10 strains) followed by P4 (6 strains). C. perfringensstrain Cp 27 has accession number ATCC-8495.

Intralytix Isolation Pathogenic PGFE ID Alpharma ID year Source Location(yes/no) Comment Type Cp1 7998B 1995 Roney Canada Yes P1 Cp2 UAZ 75 — —— — P2 Cp3 Wallers 1993 — IL Yes P3 Cp4 Pennington 1993 — IL Yes P4 Cp596-7413 1996 Roney AL Yes NT Cp6 Uaz 74 — — — — P5 Cp7 Warren 1993 — ILYes P6 Cp8 AU1 1996 — AL Yes Gangrenous P7 dermatitis Cp9 95-949 1995Fitz-Coy East Coast Yes NT Cp10 M1 2000 Fitz-Coy East Coast Yes P8 Cp11Harmes 1993 — IL Yes P3 Cp12 94-5223 1994 Thayer GA Yes P6 Cp13D00-20250 2000 Fitz-Coy MN Yes NT Cp14 UDE95-1377 1995 Fitz-Coy DE YesP9 Cp15 95-1046 1995 Fitz-Coy DE Yes Gall NT bladder Cp16 F96-01993 1996Fitz-Coy CA Yes P6 Cp17 UAZ 257 — — — — P10 Cp18 94-5228 1994 Thayer GAYes P11 Cp19 Gresbrecht A 1993 — IL Yes P6 Cp20 96-2873 1996 Roney ALYes Did not * grow Cp21 URZ298 — — — — P12 Cp22 FC1 1995 Fitz-Coy EastCoast Yes P13 Cp23 Kendall 1993 — IL Yes P4 Cp24 UDE95-1372 1995Fitz-Coy DE Yes P14 Cp25 C97M3 1997 — CO Yes P4 Cp26 Reed 1993 — IL YesP4 Cp27 AU2 1996 Roney AL Yes Gangrenous P7 dermatitis Cp28 A1A 2002Skinner DE Yes P15 Cp29 96-7414 1996 Roney AL Yes P13 Cp30 94-5230 1994Thayer GA Yes P6 Cp31 94-5224 1994 Thayer GA Yes P6 Cp32 FC2 1995Fitz-Coy East Coast Yes P4 Cp33 94-5229 1994 Thayer GA Yes P6 Cp34 7998C1995 — Canada Yes P1 Cp35 S1-1 2000 Fitz-Coy East Coast Yes P6 Cp3694-5227 1994 Thayer GA Yes P6 Cp37 Jones 1993 — IL Yes P6 Cp38 6A 2002Skinner NJ Yes P14 Cp39 S1-7 2000 Fitz-Coy East Coast Yes NT Cp40 7998A1995 Roney Canada Yes P1 Cp41 95-1000 1995 Fitz-Coy — — P4 Cp42 AU3 1996Roney — — NT

A dendrogram portraying genetic diversity of various C. perfringensstrains based on SmaI-digested PGFE patterns of C. perfringens DNA isshown in FIG. 2. Among the strains making u the 15 PFGE types, 16strains (about 46%) were grouped into PFGE types P6 (10 strains) and P4(6 strains). The remaining 20 strains clustered into eight PFGE typesrepresented by a single strain (PFGE types P2, P5, P8, P9, P10, p11, p12and p15), four PFGE type represented by two strains each (PFGE types P3,P7, P13 and P14) and one PFGE type represented by three strains (PFGEtype P1). While some of the strains within the same PFGE type wereassociated with the same geographic location/source/year of isolation(e.g., both strains in the PFGE type P3 have come from the IllinoisDisease Lab, and they both were isolated in 1995), the number of strainsin the PFGE type other than P$ and P6 was too small for makinggeneralized conclusions about their specific association with any givenfacility/location. Strains in the PFGE type P$ and P6 did not appear tobe associated with a specific location/source of isolation (e.g.,strains in the PFGE type P6 were isolated from various sources inIllinois, Georgia and California). FIG. 3 shows phage plaques producedby representative bacteriophage infecting C. perfringens strain Cp 42.

In sum, four to six candidate bacteriophages lytic for C. perfringenswere isolated on phylogenetically distinct strains from environmentalwater sources each obtained from a different poultry farm or processingplant.

Example 2 Characterization of Phages Capable of Infecting Clostridiumperfringens

The methods of Example 1 were also used for Example 2 where appropriate.

Phage Sterility: Microbial contamination was determined by (1) plating 1mL aliquots of test sample on LB agar plates and incubating replicateplates at 37° C. and 30° C. for 48 hours and (2) pre-incubating 1 mLaliquots of test sample for 37° C. for 24 hours then plating the sampleson LB agar and incubating the plates for 24 hours at 37° C. One set ofplates was incubated aerobically and another set anaerobically asindicated. Any bacterial growth at the indicated times denotescontamination.

Phage Purity: Purity of phage stocks was determined by pulsed-field gelelectrophoresis (PFGE) of uncut DNA. Approximately 100-200 ng of thephage DNA was electrophoresed in a 1% SeaKem Gold Agarose (Cambrex,Rockland, Me.) gel with 0.5× Sodium boric acid (1×SB: 10 mM sodiumhydroxide pH adjusted to 8.5 with boric acid) buffer at 14° C. in a CHEFMapper XA PFGE apparatus (Bio-Rad Laboratories, Hercules, Calif.). Therun time is 12 hours with a voltage of 6 V/cm and a linearly rampedpulse time of 0.06 s to 8.53 seconds. The gels were stained withethidium bromide and visualized with UV light.

Nucleic Acid Characterization: DNA from each batch of bacteriophage wasisolated by standard phenol-chloroform extraction method. Proteinase K(200 μg/ml) and RNAse A (1 μg/ml) were added to phage samples withtiter≧1×10⁹ PFU/ml and incubated at 37° C. for 30 minutes followed by56° C. for an additional 30 minutes. SDS/EDTA was added to a finalconcentration of 0.1% and 5 mM respectively and incubated at roomtemperature for 5 minutes. The samples were extracted once with bufferedphenol, once with phenol-chloroform and once with chloroform. Phage DNAwas ethanol precipitated and resuspended in 10 mM Tri-HCl (pH 8.0)-0.1mM EDTA (TE) buffer).

Restriction maps of the phage genomes were made by digestingapproximately 1 μg of the phage DNA with 10 units of XmnI (New EnglandBioLabs, Beverly, Mass.) according to the manufacturers'recommendations. Restriction fragments were separated on 1.0% agarosegel for 16 hours at 20V in 1× Tris-acetate-EDTA (10×TAE, EMD Chemicals)buffer and bands visualized by staining with ethidium bromide.

Protein Characterization: Phage proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, phagesamples with a titer≧1×10⁸ PFU/ml were denatured in a boiling water bathfor minutes in NuPAGE® LDS buffer fortified with DTT (Invitrogen,Carlsbad, Calif.). Aliquots were electrophoresed in a precise NuPAGE®Novex 4 to 12% Bis-Tris continuous gradient gel (Invitrogen) at 120 Vfor 110 minutes. Proteins were visualized on gels by silver-stainingusing SilverXPress® (Invitrogen) according to the manufacturers'recommendations.

Clostridium perfringens Phage Susceptibility: Forty-six C. perfringensstrains were screened for their susceptibility to the six monophages andtwo cocktails by the drop lawn method. Strains were streaked onto BHIagar plates and incubated at 37° C. anaerobically overnight. One colonyof each strain was inoculated into separate 15-ml culture tubecontaining BHI broth and incubated at 37° C. anaerobically until theOD₆₀₀ reached 0.1-0.3. One hundred microliters of each strain was mixedwith BHI soft-agar and poured onto a BHI agar plate. After the soft-agarhardened 10 μL of each phage was spotted in triplicate onto platesinoculated with C. perfringens strains. Lytic activity was observedafter overnight anaerobic incubation at 37° C.

Preparation of Phage Manufacturing Batches: Shake Flask Batches of Eachphage were carried out in 2-L flasks containing 1.5 L of BHI broth.Clostridium perfringens strains were grown in BHI broth anaerobicallyovernight at 37° C., subcultured and grown to an OD₆₀₀ of 0.1-0.3.Cultures were infected at an MOI previously determined to be optimal foreach phage (See Table 5.1.4). Growth was monitoredspectrophotometrically until lysis occurred and phage harvested byvacuum filtration (SteriCup, Millipore). Batches of each phage wereconcentrated separately and buffer exchanged with PBS by tangential flowfiltration in a Pellicon® Mini Cassette using a 50 kDa filter(Millipore).

Endotoxin Levels: Endotoxin levels for lots of phage produced weredetermined by a Limulus amoebocyte lysate (LAL) assay using achromogenic assay (QCL-1000 Kit, BioWhittaker, Walkersville, Md.)following manufacturers' recommendations. A standard curve with E. coliendotoxin (supplied with kit) ranging from 0.1 to 1.0 endotoxin units(EU) per milliliter endotoxin is constructed for each assay by plottingthe optical density at 405 nm (OD₄₀₅) versus EU/ml. Phage samples areassayed diluted in sterile water (USP, Baxter, Deerfield, Ill.) and theabsorbance at 405 nm was measured with a μQuant (Bio-Tek Instruments,Inc., Winooski, Vt.) microplate reader.

Endotoxin concentrations for each sample are calculated by linearregression from the standard curve. Standards and samples are analyzedin triplicate.

Carbohydrate Content: Carbohydrate content for all lots of phageproduced is determine by an anthrone method. A standard curve withglucose ranging from 10 to 250 μg/ml is constructed for each assay byplotting optical density at 625 nm (OD₆₂₅) versus μg/ml glucoseconcentration. Phage samples are assayed in PBS and the absorbance at625 nm was measured with a μQuant (Bio-Tek Instruments, Inc., Winooski,Vt.) microplate reader.

Carbohydrate concentrations for each sample are calculated by linearregression from the standard curve. Standards and samples are analyzedin triplicate.

Total Protein Content: The total protein content for all lots of phageproduced is determine by a bicinchoninic acid (BCA) assay using acolorometric assay (BCA™ Protein Assay kit, Pierce, Rockford, Ill.)following manufacturers' recommendations. A standard curve with bovineserum albumin ranging from 20 to 2000 μg/ml protein is constructed foreach assay by plotting optical density at 562 nm (OD₅₆₂) versus μg/mlBSA. The absorbance at 562 nm was measured with a μQuant (Bio-TekInstruments) microplate reader.

Total protein concentrations for each sample are calculated by linearregression from the standard curve. Standards and samples are analyzedin triplicate.

Electron Microscopy: High-titer phage lysates (≧10⁸ PFU/ml) werecentrifuged at 31,000×g for 2 hours and resuspended in 100 mM ammoniumacetate (pH 7.0). A drop of phage suspension was deposited on acarbon-coated Fromvar copper grid of 400 mesh. The phages werenegatively stained by adding a drop of potassium phosphotungstate (102%pH 7) and after one minute the excess fluid was withdrawn. Pictures ofthe phage particles were taken with a Phillips EM 300 transmissionelectron microscope at an acceleration voltage of 60 kV with a primarymagnification of 29,700×.

Results: Six novel bacteriophages that infect Clostridium perfringenswere isolated from environmental water or sewage sources. Each phage wascharacterized at both the protein and nucleic acid level. The optimalhost strain for propagation of each bacteriophage was identified and allwere negative for endogenous phage (Table 2).

TABLE 2 Optimal C. perfringens host strains for propagating C.perfringens-specific phages. Phage Host CPLV-42 Cp27 CPAS-7 Cp8 CPAS-12Cp26 CPTA-37 Cp27 CPAS-15 Cp8 CPAS-16 Cp42

Stocks of all six monophages and their respective host strains were madefor use in characterization and production of each phage.

PFGE analysis of uncut DNA from each of the isolated phages showed thatthey were pure monophages with genome sizes of 36 to 50 kb (FIG. 4). DNAfrom each monphage isolated was digestible with XmnI (FIG. 5). All sixof the monophages showed different protein profiled on SDS_PAGE or RFLPprofiles confirming that all six monophages are different from oneanother (FIG. 6).

Electron microscopy showed the bacteriophages to be members of theSiphoviridae or Myoviridae families of icosahedral head phages (FIG. 7)with long tails and double-stranded DNA genomes.

The ability of the six C. perfringens monophages to infect 46 C.perfringens strains is shown in Table 3. Phage CPLV-42, CPAS-16,CPTA-37, CPAS-7, CPAS-12 and CPAS-15 infected 20%, 87%, 13%, 39%, 74%and 52% of the strains screened respectively. The specificity of thephages for C. perfringens was examined by screening the susceptibilityof ten stains of E. coli, L. monocytogenes, S. enteric and P.aeruginosa. None of these 40 strains were infected by any of themonophages isolated against C. perfringens (Table 4).

TABLE 3 Susceptibility of C. perfringens strains to C.perfringens-specific bacteriophage Phage Cocktail Strain CPLV-42 CPAS-16CPAS-12 CPAS-15 CPAS-7 CPTA-37 INT-401 INT-402 ATCC + + + + + − + +13124 Cp1 − + − − − − + + Cp2 − − + + + − + + Cp3 − + + − − − + + Cp4− + + + − − + + Cp5 − + + + − − + + Cp6 − + + − − − + + Cp7 + + + + +− + + Cp8 + + − + + − + + Cp9 − + + + − − + + Cp10 − + − + − − + + Cp11− + + − − − + + Cp12 − + + − − − + + Cp13 − + + + − − + + Cp14 − + − + +− + + Cp15 + + + − − − + + Cp16 − + + + + − + + Cp17 − + + + + − + +Cp19 − + + − − − + + Cp21 − + + − − − + + Cp22 + + + + − − + + Cp23− + + + + − + + Cp24 − + + + − − + + Cp25 − + + + + − + + Cp26 − + + − −− + + Cp27 + + + − − + + + Cp28 − + − − − − + + Cp29 − + − − + − + −Cp30 − + + − − − + + Cp31 − − + − − − + − Cp32 + + − + + + + − Cp33− + + + − − + − Cp34 − − − + + + + + Cp35 − + − + + − + + Cp36 − − + + −− + + Cp37 − + + − − − + + Cp38 − + + + + − + + Cp39 − + − − − − + +Cp40 − + − − − − + + Cp41 − − + − + + + + Cp42 + + + + + + + + Cp43− + + − − − + + Cp44 − + + − + − + + Cp45 − + + − − − + + Cp46 − − − − −− − − Cp47 + + + + + + + +

TABLE 4 Susceptibility of other bacterial strains to C.perfringens-specific bacteriophage Phage Strain CPLV-42 CPAS-16 CPAS-12CPAS-15 CPAS-7 CPTA-37 Pseudomonas aeruginosa − − − Pa1 − − − − − − Pa3− − − − − − Pa7 − − − − − − Pa15 − − − − − − Pa21 − − − − − − Pa33 − − −− − − Pa42 − − − − − − Pa62 − − − − − − Pa65 − − − − − − Pa72 − − − − −− Salmonella enterica SE24 − − − − − − SS28 − − − − − − ST31 − − − − − −SHE43 − − − − − − SH49 − − − − − − S45 − − − − − − S AE 72 − − − − − −SK 103 − − − − − − SR114 − − − − − − SH162 − − − − − − Listeriamonocytogenes Lm6 − − − − − − Lm10 − − − − − − Lm23 − − − − − − Lm31 − −− − − − Lm35 − − − − − − Lm49 − − − − − − Lm62 − − − − − − Lm67 − − − −− − Lm79 − − − − − − Lm86 − − − − − − Escherichia coli Ec3 − − − − − −Ec26 − − − − − − Ec37 − − − − − − Ec41 − − − − − − Ec56 − − − − − − Ec60− − − − − − Ec65 − − − − − − Ec68 − − − − − − Ec73 − − − − − − Ec77 − −− − − −

The following strains were used to demonstrate the overall activity ofthe phage cocktail INT-401, versus a standard set of Clostridiumperfringens strains available from depositories. Strains were evaluatedfor lysis by spotting 10 microliters containing 10⁸ pfu/ml onto purelawns of each test strain spread onto BHI agar, and incubating overnightat 37° C.

TABLE 5 Strain Identifier INT-401 Depository Number Lysis ATCC 25768 +ATCC 3624 + ATCC 9856 + ATCC 3628 + ATCC 13124 + ATCC PTA-50143 + NRRLB-50143 (CP8) + NRRL B-50144 (CP26) + NRRL B-50145 (CP42) +

6 monophages were then isolated. (Table 6) Two phage cocktails INT-401(CPAS-7, CPAS-12, CPAS-15, CPAS-16 and CPLV-42) and INT-402 (CPAS-12,CPAS-15, CPAS-16 and CPLV-42), were prepared from five of the sixmonophages isolated. Table 7 gives the levels of endotoxin, totalcarbohydrate, and total protein in C. perfringens-specific cocktails.Both cocktails were effective in killing greater than 85% of the 46 C.perfringens strains screened. INT-401 was selected for use inproof-of-principle efficacy studies designed to determine the preventionof necrotic enteritis in C. perfringens challenged broiler chickens.This cocktail contains the five bacteriophages with the broadest hostrange and is likely to provide a broader (compared to INT-402) spectrumof activity in actual use against wild-type C. perfringens strains.

TABLE 6 Summary of C. perfringens monphage batch preparation Phage HostStrain MOI Culture Time (h) Titer (PFU/ml) CPLV-42 Cp27 1 5 5 × 10¹⁰CPAS-16 Cp42 1 3 1 × 10⁸ CPAS-12 Cp26 1 4-5 1 × 108 CPTA-37 Cp27 1 4.5 4× 10⁸ CPAS-15 Cp8 1 4-5 5 × 10⁸ CPAS-15 Cp8 1 4-5 6 × 10⁹

TABLE 7 Levels of endotoxin, total carbohydrate, and total protein in C.perfringens-specific cocktails. Content INT-401 INT-402 Endotoxin(EU/ml) 384 288 Total Carbohydrate 170 43 (μg/ml) Total protein 661 175(μg/ml)

Example 3 Protocol for Bacteriophage Treatment as Therapy or Preventionof Necrotic Enteritis in Broiler Chickens Challenged with Clostridiumperfringens

Broiler Chickens: A total of 576 male day-old broiler chickens wereassigned to treatment on day 0. There were no vaccinations (Mareks orBronchitis) or antibiotics applied to eggs or chicks at the hatchery.

Housing: The 64-pen broiler chicken research facility at maple leafAgresearch was used to conduct the study. Forty-eight pens, eachproviding approximately 10 square feet of floor space, were assigned totreatment groups. Each pen had a concrete floor and nylon meshpartitions supported by PVC frame. Adjacent pens were separated by asolid 12-inch high plastic barrier at bird level. Each pen waspermanently identified by number and contained 12 birds on day 0. Thebarn was heated by two natural gas heaters, which were equally spacedand positioned to warm incoming air at the south wall of the building.Air was exhausted by fans located on the north-facing wall of thebuilding. Each pen contained one nipple-type drinker, which providedclean drinking water ad libitum. Water was de-chlorinated. Dry feed wasprovided ad libitum in trough-type feeders (one per pen) of 5-kgcapacity. New wood shavings were used as bedding.

Management: Lighting program, barn temperature, litter type and othermanagement practices were typical of commercial broiler chickensproducers in the local geographic area and is fully documented in theraw data. Birds, which were moribund and unable to reach food or water,were culled and euthanized by carbon dioxide gas.

Bodyweight, pen number, date of death and cause of death were determinedby necropsy and recorded for each bird culled or found dead during thestudy.

Experimental design: A randomized complete block design was used tostudy the effects of eight treatments. The treatments were as follows:

TABLE 8 Treatment design Treatment code C. perfringens challenge TestArticle 1 No No 2 Yes No 3 Yes Yes 4 Yes Yes 5 Yes Yes 6 Yes Yes 7 YesYes 8 Yes Yes

There were 8 pens per block (8 treatments) and 6 blocks (replicates) fora total of 48 pens. (See Section 4.2, Deviation #2 for change to abovetreatment to block assignment).

Treatment Groups:

Treatment 1—Control UUC (no C. perfringens challenge or bacteriophageadministration)

Treatment 2—Control IUC (C. perfringens challenge or withoutbacteriophage cocktail)

Treatment 3—In ovo injection of phage cocktail at day 18 incubation.

Treatment 4—Spray application of phage cocktail to chicks afterhatching.

Treatment 5—In ovo injection of phage cocktail at day 18 incubation andspray application of phage cocktail to chicks after hatching.

Treatment 6—In ovo injection of phage cocktail at day 18 incubation,spray application of phage cocktail to chicks after hatching and oralgavage of bacteriophage cocktail on Day 7 through 13.

Treatment 7—Bacteriophage cocktail administered via oral gavage from Day7 through 13 (oral gavage ceased on the day of Clostridium perfringenschallenge).

Treatment 8—Bacteriophage cocktail administered via oral gavagebeginning on Day 14 (concurrent with Clostridium perfringens challenge)through Day 18.

Feeding Program: The following feeding program was used in the study:

TABLE 9 Feeding Program Formulation Day Feed Type Number  0-13 9:00 p.m.Day 13 to 9:00 a.m. Starter 282 Day 14 14-21 None. Feed was withdrawnNone Starter 282

Feed Sampling: The investigator's representative was present during feedmanufacture. Ten representative samples were taken from each batch offinal feed, composited and divided into three samples for proximateanalysis, and retainer samples, respectively.

Administration of Clostridium perfringens challenge: A Clostridiumperfringens isolate originating from a field case of necrotic enteritisin Ontario was used in the study. Inoculums contained approximately 10⁸cfu Clostridium perfringens per mL at time of feeding. Feed waswithdrawn from all birds for approximately 8 hours prior to firstintroduction of challenge. Inoculum was administered to birds via feedin the afternoon and night commencing Day 14 P.M. and ending Day 15 A.M.using trough-type feeders. A suitable quantity of assigned feed(approximately 0.150 kg) and an amount of inoculum equal toapproximately 1.5 times the weight of feed was added to each feeder.When this procedure was complete for all pens assigned to the challenge,feeders were returned to their corresponding pen. Inoculum-feed mixtureremaining at the end of the half-day period was weighed and discarded.

Lesion scoring of sacrificed birds: Three birds were randomly selectedfrom each pen on Day 16 and euthanized. These birds were scored grosslyfor necrotic enteritis and coccidiosis lesions:

TABLE 10 Necrotic enteritis scoring Necrotic enteritis score Description0 Normal, no evidence of gross lesions 1 Thin, friable small intestine 2Focal necrosis and/or ulceration 3 Patchy necrosis 4 Severe extensivenecrosis (typically seen in birds which have died from NE)

Clostridium perfringens culture of small intestine segment: A smallintestinal segment was collected from 40 birds that died on or after Day15 and had a gross diagnosis of necrotic enteritis. The segment wasforwarded to the Department of Pathology at the University of Guelph forC. perfringens culture. Culture results were reported as positive ornegative for C. perfringens. Samples of positive bacterial cultures wereforwarded to Intralytix for testing for phage susceptibility. Inaddition, 144 ileum samples were collected from birds sacrificed for C.perfringens lesion scoring on Day 16. These samples were quantitativelytested at the above referenced laboratory and microbiological sampleswere forwarded to Intralytix for additional characterization of phageactivity.

Necropsy: All birds that died or were euthanized were submitted to thestudy pathologist for gross necropsy to determine the cause of death.

Observation and calculation of variables:

-   -   1) Bodyweight and number of birds per pen on Days 0, 14, and 21.    -   2) Amounts of each feed consumed by each pen.    -   3) Bodyweight and date of death for birds which were culled or        died    -   4) Feed conversion was calculated on a pen basis as feed        consumed/[total weight of live birds+total weight of dead and        culled birds+total weight of sacrificed birds] for the 0-14,        14-21 and 0-21 Day periods.    -   5) Average bodyweight per pen was calculated as a total weight        of live birds at the time of weighing/number of live birds at        the time of weighing.    -   6) Daily feed intake (grams) per live bird day was calculated on        a pen basis for Day-14, Day 14-21 and Day 0-21.    -   7) Apparent cause of death was recorded for all birds that died        or were culled. Total mortality and mortality from necrotic        enteritis will be calculated on a pen basis.    -   8) Evaluation of the effects of the in ovo injection treatments        on percent hatch and chick health at the hatchery    -   9) Necrotic enteritis lesion score of sacrificed birds (Day 16).    -   10) Birds were observed on a flock basis at least once daily and        observations were recorded.

Test substance disposition: Remaining bacteriophage cocktail testsubstance was destroyed by incineration and destruction is documented inthe study records.

Bird disposition: Birds (treated and control) were humanely euthanizedat the end of the study and disposed of via incineration and method anddate of disposition was recorded in the study records. Hatchery wasteand unused in ovo bacteriophage injected eggs were disposed of viaincineration. Hatched chicks that had been in ovo injected or sprayedwith bacteriophage but not assigned to the study were humanelyeuthanized and disposed of via incineration.

Original data: Original data is submitted to the sponsor together withthe final report. An exact copy of the final report and data will bemaintained at Maple Leaf Agresearch for a minimum of two years.

Documentation: All raw (original) data was recorded in black ink on datasheets bearing the trial number. Corrections were made by drawing asingle line through the original entry and writing the correct entrybeside it together with initials of the person making the correction,the date the correction was made and the reason for the correction.Defined error codes were used to record the reason for correcting a datapoint.

Statistical analysis: Randomized complete block design was to be used.Pen location within the facility was the blocking factor. The pen wasthe experimental unit for statistical analysis. A one-way treatmentstructure was utilized with each treatment being replicated six times(once within each block, except as detailed in Deviation #2, Section4.2). Mortality data was transformed prior to analysis of variance.Mixed models analysis was used to analyze all data. Means were comparedusing an appropriate multiple range test.

Amendment #1: This amendment clarifies dates, eliminated vaccineadministration and any potential interference vaccine might have withthe Test Article, and detailed exact doses of Test article to beadministered during “in ova” injection (0.2 mL), spraying (about 7 mLper 100 chicks) and oral dosing (0.5 mL per bird per day).

Amendment #2: This amendment redefined the dosage of “in ovo”administration (0.05 mL per egg), spraying (about 7 to 22 mL per 100chicks). The upper range of 22 mL was actually used for the spray.

Deviations: Two deviations occurred and are described in the Protocolsection of the Study Binder. A brief description follows:

Deviation #1: Only 12 birds were assigned to pens instead of 15described in the protocol. This will have an impact on the statisticalpower of the study, particularly the mortality data.

Deviation #2: Block 3 was assigned 2 treatment pens and no treatment 5pen caused an imbalance in the design. Least square means will bereported to correct for the unequal representation per treatment group.This is not expected to have a major influence on the power of thestudy.

Example 4 Results for Bacteriophage Treatment as Therapy or Preventionof Necrotic Enteritis in Broiler Chickens Challenged with Clostridiumperfringens

The results of this study are summarized in Tables 10 to 12. A detailedstatistical analysis was performed.

Hatchery: “in ovo” injection of eggs with test Article (0.05 mL per egg)for Treatments 3, 5 and 6 was performed at 18 days of incubation usingan Embrex machine and followed the standard industry protocol with thefollowing exceptions: Marek's vaccine and antibiotic (Excenel) were notincluded. The standard procedure also involved applying a small amountof chlorine solution over the injection hole just post injection. Forthis trial, 1259 fertile eggs were transferred without being injectedand hatched at 96.6%. The 775 fertile eggs injected with Test Articlehatched at 95.5%.

TABLE 11 Delivery Routes of Bacteriophage on weights of broiler chickenschallenged with necrotic enteritis. Average live weights (kg)²Treatment¹ Day 0 Day 14 Day 21 Day 35 Day 42 Control .046 .330 .750^(A)1.917^(A) 2.776^(A) Challenged control .046 .340 .618^(C) 1.530^(C)2.342^(C) BMD control .045 .340 .634^(C) 1.795^(B) 2.686^(AB) Gavagedphage .045 .328 .641^(C) 1.762^(B) 2.601^(B) Phage in water .046 .348.694^(B) 1.812^(AB) 2.664^(AB) Phage in feed .045 .333 .658^(BC)1.754^(B) 2.592^(B) SEM .000 .010 .018 .045 .059 Pr > F .5309 .7492.0003 .0001 .0004 ¹LSMEANS were provided for each treatment. Thetreatment group included a control. Challenged control, BMD 50 g/ton asa medicated control, oral gavaged phage, phage provided via water andphage provided via feed. The bacteriophage used was Intralytix C.perfringens Phage Cocktail - 4.8 × 10⁹ pfu/ml. On Day 14, all the birdswere orally inoculated with a coccidial inoculums containingapproximately 5,000 oocysts of E. maxima per bird. All groups werechallenged with Clostridium perfringens on Days 18, 19, and 20. OralAdministration of phage cocktail via gavage drinking water and feedapplication will occurred on days 17, 18, 19, 20, and 21. ²Standarderror of the LSMEANS ^(A,B,C)Means within columns with differentsubscripts are significantly different.

Three treatments (#'s 4, 5 and 6, Table 10) were also sprayed with TestArticle at the hatchery after hatch. A commercial spray cabinet designedfor administering coccidiosis vaccine was used to deliver the TestArticle at a rate of 22 mL per box or approximately 0.22 mL per bird.These birds were held in the hatchery for an extra ½ hour to permitdrying to transport to the research farm.

Challenged pens were provided with 1.66 kg of Clostridium perfringensinoculums/feed mixture and all consumed at least 1.25 kg except one, achallenged control pen. This pen suffered from severe water restrictiondue to a technical problem and for this reason was removed from theanalysis. Due to the deviation described above the challenged control(Treatment 2) was assigned one extra pen and Treatment 5 one less pen.With this slight imbalance in design, least squares means are reported.There was no significant (p>0.05) difference between challenged groupsin quantity of inoculum consumed.

The primary criteria for evaluating the effectiveness of Test Articleand its method of administration is mortality attributable directly toClostridium perfringens challenge. No birds died from Necrotic Enteritis(NE) in the non-challenged control (Treatment 1) and this wassignificantly (p<0.01) different than the challenged control with 25.9%of the birds in a pen dying of NE. Birds treated (Treatment 8) by OralGavage (OG) from the day of challenge (day 14) until day 18 had thelowest mortality (5.6%) of the phage treated groups and this was notsignificantly (p>0.05) different from the non-challenged control(Treatment 1). Two of the “in ovo” groups (Treatment 3 and 6) hadintermediate NE mortality, which was not significantly (p>0.05)different from either Control groups (Treatments 1 and 2).

Three birds per pen were sacrificed at Day 16 to detect lesions typicalof NE and to determine the presence of Clostridium perfringens in eithera defined segment (approximately 3 to 4 cm distal to the duodenum) ifnot lesions were present or a segment surrounding an identified lesion.Although not significantly (p>0.05) different from the other treatmentgroups, there were no “typical” NE lesions found in the non-challengedcontrol (Treatment 1). No significant (p>0.05) difference betweentreatments was found for lesion scores.

Clostridium perfringens (Cp) bacterium were isolated from all groupsincluding the non-challenged control. We do not know if the strainisolated from the non-challenged control was the same as the challengedstrain. However, Treatment 1 was numerically lower for Bacterial Scoresfor Cultures and this was consistent with the significantly (p<0.05)lower scores (Table 1) for Smears. No other trends were evident in theBacterial Score means for either Cultures or Smears between the othertreatment groups.

TABLE 12 Delivery Routes of Bacteriophage on weight gains of broilerchickens challenged with necrotic enteritis Average live weights (kg)²Day Day Day Treatment¹ Day 0-14 Day 0-21 14-21 0-35 0-42 Control .284.705^(A) .421^(A) 1.871^(A) 2.730^(A) Challenged control .294 .572^(C).278^(C) 1.484^(C) 2.296^(C) BMD control .295 .589^(C) .294^(C)1.750^(B) 2.641^(AB) Gavaged phage 283 .596^(C) .313^(BC) 1.716^(B)2.556^(B) Phage in water .302 .648^(B) .346^(B) 1.766^(AB) 2.618^(AB)Phage in feed .287 .612^(BC) .325^(BC) 1.709^(B) 2.547^(B) SEM .010 .018.014 .045 .059 Pr > F .7559 .0003 .0001 .0001 .0004 ¹LSMEANS wereprovided for each treatment. The treatment group included a control.Challenged control, BMD 50 g/ton as a medicated control, oral gavagedphage, phage provided via water and phage provided via feed. Thebacteriophage used was Intralytix C. perfringens Phage Cocktail - 4.8 ×10⁹ pfu/ml. On Day 14, all the birds were orally inoculated with acoccidial inoculums containing approximately 5,000 oocysts of E. maximaper bird. All groups were challenged with Clostridium perfringens onDays 18, 19, and 20. Oral Administration of phage cocktail via gavagedrinking water and feed application will occurred on days 17, 18, 19,20, and 21. ²Standard error of the LSMEANS ^(A,B,C)Means within columnswith different subscripts are significantly different.

There was a significant (p<0.05) higher chick weight for one of thegroups (Treatment 4) receiving Test Article by Spray at the hatchery.This was not significantly different than one (Treatment 5) of the othertwo groups receiving the Spray. This may be the result of theseTreatments retaining more moisture from the Spray procedure.Pre-challenge, on Day 14, there were no significant differences (p>0.05)in body weight between Treatments. After challenge, at Day 21, thenon-challenged control (Treatment 1) was significantly (p<0.05) heavierthan the birds receiving Test Article by Oral Gavage (Treatment 7) priorto challenge. No other differences in growth performance were detected.

Total mortality in the non-challenged control was high at 13.9%. Asindicated in the necropsy records much of this non-NE mortality was dueto internal infections, omphalitis (yolk sac infections) and suddendeath. This high early chick mortality is not typical. Total mortalitywas significant (p<0.05) lower for the non-challenged control (Treatment1, 13.9%) and the birds receiving Oral Gavage from day 14 to day 18(Treatment 8, 12.5%) than Treatment 5 (37.0%).

TABLE 13 Delivery Routes of Bacteriophage on feed conversion of broilerchickens challenged with necrotic enteritis Feed conversion ratio (feedto gain) Day Day Day Treatment¹ Day 0-14 Day 0-21 14-21 0-35 0-42Control 1.703 1.532^(D) 1.417^(C) 1.709^(D) 1.892^(D) Challenged control1.600 1.912^(A) 2.284^(A) 2.483^(A) 3.226^(A) BMD control 1.5611.892^(AB) 2.130^(A) 2.077^(B) 2.652^(B) Gavaged phage 1.662 1.760^(BC)1.864^(B) 1.814^(CD) 2.086^(C) Phage in water 1.562 1.676^(C) 1.778^(B)1.813^(CD) 2.066^(C) Phage in feed 1.680 1.777^(ABC) 1.868^(B) 1.841^(C)2.089^(C) SEM² .045 .051 .081 .047 .039 Pr > F .1359 .0002 .0001 .0001.0001 ¹LSMEANS were provided for each treatment. The treatment groupincluded a control. Challenged control, BMD 50 g/ton as a medicatedcontrol, oral gavaged phage, phage provided via water and phage providedvia feed. The bacteriophage used was Intralytix C. perfringens PhageCocktail - 4.8 × 10⁹ pfu/ml. On Day 14, all the birds were orallyinoculated with a coccidial inoculums containing approximately 5,000oocysts of E. maxima per bird. All groups were challenged withClostridium perfringens on Days 18, 19, and 20. Oral Administration ofphage cocktail via gavage drinking water and feed application willoccurred on days 17, 18, 19, 20, and 21. ²Standard error of the LSMEANS^(A,B,C,D)Means within columns with different subscripts aresignificantly different.

TABLE 14 Delivery Routes of Bacteriophage on mortality and lesion scoresof broiler chickens challenged with necrotic enteritis Mortality (%)²Necrotic Total include all causes Necrotic Enteritis Day Day DayEnteritis lesion Treatment¹ 0-21 0-35 0-42 Day 0-42 scores³ Control2.67^(CD) 2.67^(D) 4.00^(D)  0^(D)  0^(B) Challenged control 41.33^(A)66.00^(A) 66.67^(A) 64.00^(A)  .9^(A) BMD control 24.67^(B) 51.33^(B)53.33^(B) 50.00^(B) 1.1^(A) Gavaged phage 10.00^(C) 16.67^(C) 18.00^(C)14.00^(C)  .1^(B) Phage in water .67^(D) 67^(D) 3.33^(D)  0^(D)  .1^(B)Phage in feed 2.00^(CD) 3.33^(D) 5.33^(D)  .66^(D)  .4^(B) SEM⁴ 2.972.71 2.81  2.76  .2 Pr > F .0001 .0001 .0001  .0001  .0006 ¹LSMEANS wereprovided for each treatment. The treatment group included a control.Challenged control, BMD 50 g/ton as a medicated control, oral gavagedphage, phage provided via water and phage provided via feed. Thebacteriophage used was Intralytix C. perfringens Phage Cocktail - 4.8 ×10⁹ pfu/ml. On Day 14, all the birds were orally inoculated with acoccidial inoculums containing approximately 5,000 oocysts of E. maximaper bird. All groups were challenged with Clostridium perfringens onDays 18, 19, and 20. Oral Administration of phage cocktail via gavagedrinking water and feed application will occurred on days 17, 18, 19,20, and 21. ²Standard error of the LSMEANS ³On Day 22, scoring was basedon a 0 to 3 score, with 0 being normal and 3 being the most severe.⁴Standard error of the LSMEANS ^(A,B,C,D)Means within columns withdifferent subscripts are significantly different.

The non-challenged control (Treatment 1) had the numerically highest(102 grams per bird per day) feed intake and this was significantly(p>0.05) more than Treatment 5 (84 grams per bird per day). Although themeans comparison was not significant (p>0.05), Treatment 8 had thenumerically best FCR (1.477) of the Phage treated groups and equal tothe performance of the non-challenged control.

Conclusions:

-   -   1. A successful Clostridium perfringens (Cp) challenge was        achieved. The positive control had 25.9% of the birds die from        Necrotic Enteritis compared to the negative control (0.0%).    -   2. Oral Gavage of Test Article to birds on the day of challenge        (Day 14) and for the next four days significantly reduced        mortality due to NE. Growth performance in this group was        numerically equivalent to the Non-Challenged control.    -   3. Two of the three “In ovo” treatments had numerically reduced        NE mortality (9.6 and 14.8%) when compared to the Challenged        control (25.9%).    -   4. Oral Gavage of Test Article prior to challenge was        ineffective in preventing NE mortality due to Cp challenge.    -   5. Spray of Test Article to chicks at the hatchery did not        significantly (p>0.05) reduce NE mortality from Cp challenge.    -   6. The precision of this trial was reduced by several factors        including fewer birds being assigned to pens at day old than        specified in the protocol and high early non-challenged related        mortality.

Example 5 Sequence Analysis of C. perfringens Bacteriophage

Media: Brain Heart Infusion (BHI) broth or BHI agar supplemented with250 mg/L cycloserine was used to grow all C. perfringens isolates.Luria-Bertani (LB) broth and LB agar was used to grow all aerobicstrains. All media were obtained from EMD Chemicals, Gibbstown, N.J.

Microoganisms: Clostridium perfringens strains were from Intralytix Inc.Culture Collection, Baltimore, Md. As part of the collection process,isolates were checked for purity and frozen at −80° C. in 30% glycerol.Most of the work was performed in an anaerobic chamber (Plas-Labs,Lansing, Mich.), that contained a 90% N₂-5% H₂-5% CO₂ atmosphere.Escherichia coli, Listeria monocytogenes, Salmonella enteric, andPseudomonas aeruginosa strains were from the Intralytix Inc. CultureCollection and all were grown aerobically.

Bacteriophage: All bacteriophage were isolated from environmental watersources.

Phage DNA Isolation: DNA from each batch of bacteriophage was isolatedby standard phenol-chloroform extraction method. Proteinase K (200μg/ml) and RNAse A (1 μg/ml) were added to phage samples withtiter≧1×10⁹ PFU/ml and incubated at 37° C. for 30 minutes followed by56° C. for an additional 30 minutes. SDS/EDTA was added to a finalconcentration of 0.1% and 5 mM respectively and incubated at roomtemperature for 5 minutes. The samples were extracted once with bufferedphenol, once with phenol-chloroform and once with chloroform. Phage DNAwas ethanol precipitated and resuspended in 10 mM Tri-HCl (pH 8.0)-0.1mM EDTA (TE) buffer.

Phage Sequencing: The DNA from each of the phages was sequenced usingstandard automated sequencing methods.

Sequence Analysis: To identify the predicted open reading frames (ORFs)WPA uses a combination of CRITICA (1) and GLIMMER (2). The results fromthese programs are combined and the optimal reading frames are extractedfrom the combined data set.

Each of the two programs uses different algorithms for identifying openreading frames, and each has its benefits and drawbacks. However, bycombining the output from both tools WPA is able to optimize thepredicted ORFs that they can identify.

WPA uses an automated annotation system in which assignments aregenerated primarily by sequence similarity searches between the de novoindentified ORFs and other ORFs in their databases. In this case, theBLAST algorithm was used to compare predicted protein sequences for theannotation. In addition to the publicly available databases forcomparison WPA has a phage database that contains approximately 400phage genomes which they feel represents the best phage datasetavailable.

Following the automated annotation and assignment phase, the assignmentsfor each genome are manually curated by Intralytix to see if any of the17 undesirable genes (Table 15) are present.

TABLE 15 List of undesirable genes encoded in the bacteriophage genomesToxin and its Encoding Gene Bacterial Pathogen Enterotoxin A (entA)Staphyloccocus aureus Enterotoxin A (sea, sel) StaphyloccocusEnterotoxin A (sea) Staphyloccocus aureus Staphylokinase (sak)Staphyloccocus aureus Enterotoxin P (sep) Staphyloccocus aureusExfoliative toxin A (eta) Staphyloccocus aureus Diptheria toxin (tox)Corynebacterium diptheriae Shiga toxins (stx1, 2) Escherichia coliCytotoxin (ctx) Pseudomonas aeruginosa Cholera toxin (ctxA) Vibriocholerae Cholera toxin (ctxB) Vibrio cholera Zona occludens toxin (zot)Vibrio cholerae Neurotoxin (C1) Clostridium botulinum Enterohaemolysin(hly) Escherichia coli Streptococcal enterotoxin A (speA) Streptococcuspyrogenes Streptococcal enterotoxin C (speC) Streptococcus pyrogenesStreptococcal enterotoxin K(speK) Streptococcus pyrogenes

Five of the six phages were sequenced. The sequences of each of the fivephage genomes were obtained and each predicted open reading frameidentified in each genome (Table 16). Each of the predicted genes wasannotated. None of the 17 undesirable genes (Table 15) were found in thegenomes of any of the five phages for which sequences were available(Table 17).

TABLE 16 Number of predicted ORFs for each C. perfringens-specificbacteria. Phage Number of Open Reading Frames (ORFs) 1 67 2 77 3 69 4 475 67

Example 6 Use of a Water-Conditioning Agent in the Phage Cocktail

Phage cocktail INT-401 at a final concentration of 1×10⁷ pfu/ml wasplaced into treated (containing 50 mM citrate-phosphate-thiosulfate(CTP) buffer, comprising about 40 mg sodium thiosulfate, 6.0 gm disodiumphosphate (anhydrous), 1.1 gm citric acid (anhydrous) per liter ofdeioinized water, pH 7.0 (added at a 1:10 ratio to water) and untreated(distilled water) solutions containing bleach at the levels indicated inTable 18, and allowed to stand for one hour at room temperature. Sampleswere taken, and 10 microliters were spotted onto BHI agar mediumcontaining lawns of C. perfringens ATCC 13124 and allowed to dry. Plateswere incubated overnight at 37° C., and phage inactivation was scored bythe absence of a lytic clearing zone visible on the bacterial lawn.

Results: The results in Table 18 demonstrated the thiosulfate-containingbuffer was able to protect phage cocktail INT-401 against oxidation dueto chlorine bleach exposure. This conditioning agent could therefore beapplied to chlorinated water as a means to allow the phage cocktail toretain activity in a commercial poultry watering system.

TABLE 18 Water Conditioning Agent Allowing Protection of Phage CocktailINT-401 in the Presence of Hypochlorite Lysis Response vs. ATCC 13124Hypochlorite concentration Conditioned water Unconditioned water 0 ++ ++0.5 ++ + 1.0 ++ + 2.0 + − 4.0 + − 6.0 + −

Example 7 Treatment of C. septicum with INT-401

Seven C. septicum and two C. perfringens turkey isolates, associatedwith turkey dermatitis or related turkey disease complexes were receivedfrom Marion Morgan, University of Arkansas Dept. of Poultry Science, inmeat broth. Strain ATCC 13124 (C. perfringens) was used as an internalcontrol and was used from a frozen source from the Alpharma AnimalHealth Culture Collection, Chicago Heights, Ill. Strains weresub-cultured; 0.1 ml of broth into 9 ml Wilkins-Chalgren medium, andWilkens-Chalgren and supplemented Mueller-Hinton agar, followed byovernight growth at 37° C. Once colonies were observed, at least 10representative colonies of each strain were transferred into 1 mlsterile saline, to a uniform turbidity corresponding to about 10⁸-10⁹cfu/ml cell density. The saline suspensions were inoculated at 0.1 mlper plate and evenly distributed to one plate each of unsupplementedWilkins-Chalgren and Mueller-Hinton agar (supplemented with 0.25% w/vglucose). Commercial antibiotic disks were then placed onto the surfaceof each plate, along with 15 μl spots of INT-401 phage cocktail, (ca.10⁸ pfu phage/ml). The spots were allowed to completely absorb onto theplate surface. All plates were then incubated at 37° C. in an anaerobicvessel (using BBL Gas-Pak® to generate anaerobiosis). Zones ofinhibition were measured (in millimeters) for each antibiotic, andevaluation of lysis of bacteria due to INT-401 phage was recorded foreach strain. Table 17 summarizes the results of the study.

TABLE 17 Lysis response of Clostridium spp. to Phage Cocktail INT-401 onTwo Culture Media Formulations. Lysis from Lysis from INT-401 INT-401Strain Species (Wilkins-Chalgren) (Mueller-Hinton) 08-114 C.septicum + + 08-121 C. septicum − (+) 08-126 C. septicum (+) − 08-183 C.septicum (+) (+) 08-196 C. septicum (+) (+) 08-205 C. septicum (+) −09-03 C. septicum + + 08-41 C. perfringens + + 08-146 C. perfringens + +ATCC 13124 C. perfringens + + (control) Responses scores: + clear lysisof entire challenge spot; − no lysis observed; (+) partial lysis (clearindividual plaques or less turbid zone observed).

The results show that disease-associated poultry C. septicum strains maybe lysed by phage cocktail INT-401. In this case 2/7 strainsdemonstrated clear-cut lysis comparable to C. perfringens controls, andall 7 strains showing some response to the product. Some differencesbetween culturing medium were noted.

Example 8 Bacterial Lysis Using Both Phage and Antibiotic Treatments

Antibiotic enhancement of phage lysis: Lawns of overnight growth of hoststrain CP8, lysed by 4/5 component phages of INT-401, were streaked ontoBHI and Shaedler agar medium, and at least 3 10 μl spots of −1, and −2dilutions of INT-401 phage were placed onto the surface, and allowed todry. Subsequently, commercial disks of bacitracin (10 microgram, HardyDiagnostics) and chlortetracycline (30 microgram, Hardy Diagnostics)were placed adjacent to the phage spots at a distance of 1-3 mm. Controlspots and control antibiotic disks were placed >15 mm apart. The plateswere then incubated overnight at 37° C., in a commercial anaerobic jar,and data were recorded for zones of inhibition present/absent andinteractive effects, if any.

Strain CP-8 demonstrated an enhanced lysis effect at the intersection ofINT-401 plus antibiotics chlortetracycline or bacitracin. The on-plateregions of overlapping interaction of both phage and antibiotic againstthe Clostridium bacteria lawn, had somewhat larger (by 1-3 mm) clearingareas, with no visible encroaching bacterial growth on the edges, havinga distinctive and superior clearing zone with clean, straight edges. Thecontrol spots or disks had visibly or microscopically less distinctivelyclear edges, and occasional visible single colonies encroaching on theedges (these have borderline, lower concentrations present thus willallow some growth of some bacteria). This result shows that the phagecocktail product INT-401 and antibiotics, both of which are inhibitoryof Clostridium spp. (and in particular Clostridium perfringens) and forantibiotics which are approved and are routinely used for growthpromotion, disease prevention, disease control, or disease treatmentpurposes, have demonstrably compatible activities. Both phage andantibiotics can kill or inhibit bacteria by different mechanisms andmodes of action. This test shows to a significant degree that the twomechanisms do not interfere with each other and appear to providecomplementary killing activity. Multiple antibiotics and phage mixtureshave at least an additive effect and in several cases show synergisticactivities against bacteria, using qualitative or quantitativemethodology

Example 9 INT-401 Treatment Versus Antibiotic Resistant C. perfringensStrains

Antibiotic-Resistant Clostridium perfringens test: Strains of C.perfringens, were grown and subsequently challenged, using spots (−1 or−2 dilutions) of phage cocktail INT-401, according to standardizedmethods on BHI agar medium. Both antibiotic-sensitive controls (ATCCreference and host strains), as well as animal-origin strains in theAlpharma Animal Health culture collection were tested. The strains wereidentifiable by previously measured, notable antibiotic resistances: 10pan-susceptible strains (sensitive to all tested antibiotics), 6bacitracin-tolerant strains; of which 3 had MIC>50 ppm, and 2macrolide-resistant (MIC>32 for tylosin tartrate) strains. Dataconcerning INT-401 lysis were recorded based on observation of visiblelysis on lawns following overnight incubation. Table 17 summarizes theresults, showing lysis patters versus examples of antibioticpan-susceptible (control), and specific antibiotic-resistant animalstrains of C. perfringens.

TABLE 17 Lysis response of Antibiotic-tolerant or -sensitive, animalClostridium perfringens strains to Phage Cocktail INT-401. DefinedAntibiotic Resistance Lysis from INT-401 Strain (MIC, ppm) (BHI agar)ATCC 3624 bac. tyl. (<2), S + ATCC 13124 bac. tyl.(<2), S + (control)ATCC 9856 bac. tyl. (<2), S + Warren bac (50) + G84239 bac (4) + TO4213bac.(2) − M3836 bac.(4) + CL3801 bac.(50) − EV3839 bac.(50) + N84236 tyl(≧32) + CH4225 tyl (≧32) + JGS4029 bac. tyl.(<2), S − JGS4027 bac.tyl.(<2), S + JGS4026 bac. tyl.(<2), S + JGS4016 bac. tyl.(<2), S +JGS4057 bac. tyl.(<2), S − JGS4048 bac. tyl.(<2), S − JGS4036 bac.tyl.(<2), S + S = Pan susceptible to all tested antibiotics.bac-bacitracin, tyl, tylosin (and other macrolide cross-resistant classantibiotics including lincomycin and erythromycin)

In summary, 7/10 of pan-susceptible strains, compared with 4/6 totalbacitracin resistant (2/3 highly bacitracin-resistant), and 2/2 highlymacrolide-resistant (tylosin-resistant) strains were lysed by theINT-401 cocktail challenge. The results show that phage can lyseantibiotic-resistant bacteria to the same or greater degree asantibiotic-susceptible types.

The terms “first”, “second”, and the like herein, do not denote anyorder, quantity, or importance, but rather are used to distinguish oneelement from another, and the terms “a” and “an” herein do not denote alimitation of quantity, but rather denote the presence of at least oneof the referenced item. All ranges disclosed herein are inclusive andcombinable.

While the invention has been described with reference to a preferredembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims.

All cited patents, patent applications, and other references areincorporated by reference in their entirety.

TABLE 17 Annotations of all predicted genes for each C.perfringens-specific bacteriophage genome Gene ID Annotated FunctionCPAS-7  1 Presumed portal vertex protein  2 Ring-infected erythrocytesurface antigen  3 Hypothetical protein  4 Hypothetical protein  5FKBP-type peptidyl-prolyl cis-trans isomerase (trigger factor)  6Isocitrate dehydrogenase kinase/phosphatase  7 Phage-like element PBSXprotein xkdH  8 Phase 1 flagellin  9 Type I restriction-modificationsystem restriction subunit (EC 3.1.21.3) 10 Sarcosine oxidase, alphasubunit 11 Hypothetical protein 12 Phage-like element PBSX protein xkdK13 Phage-like element PBSX protein xkdM 14 Hypothetical protein 15Hypothetical protein 16 Phage protein 17 Hypothetical protein 18Phage-like element PBSX protein xkdQ 19 Ribosomal protein S4 and relatedproteins 20 Phage-like element PBSX protein xkdS 21 Hypothetical protein22 Phage-like element PBSX protein xkdT 23 Tail fiber 24 Heat shockprotein 90 25 Hypothetical protein 26 Bacteriocin uviB precursor 27N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) 28 ABC transporter,permease protein 29 No hits 30 Transposase 31 DNA repair protein RadA 32Transcriptional regulator 33 Hypothetical protein 34 Hypotheticalprotein 35 Thymidine kinase (EC 2.7.1.21) 36 Tryptophanyl-tRNAsynthetase (EC 6.1.1.2) 37 CMP-binding factor 38 3-isopropylmalatedehydratase large subunit (EC 4.2.1.33) 39 DNA ligase 40 Putativepenicillin-binding protein 41 Signal transducer and activator oftranscription 1 42 Chemotaxis protein CHED 43 Gramicidin S synthetase I(EC 5.1.1.11) 44 ABC transporter ATP-binding protein 45 PutativeATP-dependent DNA helicase 46 DNA polymerase I 47 Hypothetical protein48 Hypothetical protein 49 Phenylalanyl-tRNA synthetase beta chain 50Terminase large subunit 51 Terminase small subunit 52 Hypotheticalprotein 53 CobT protein 54 Putative chromosome segregation protein, SMCATPase superfamily 55 Deoxycytidylate deaminase (EC 3.5.4.12) 56 aceE;pyruvate dehydrogenase e1 component oxidoreductase protein 57Hypothetical protein 58 Hypothetical protein 59 Aspartic acid-richprotein aspolin2 60 CDEP 61 Nucleolin 62 Peptide ABC transporter,ATP-binding protein 63 GTP-binding protein SAR1 64 gp56 dCTPase 65Hypothetical protein 66 Homeobox-leucine zipper protein 67 DNA repairprotein recN CPAS-16  1 Developmentally regulated GTP-binding protein 1 2 Presumed portal vertex protein  3 Ring-infected erythrocyte surfaceantigen  4 Hypothetical protein  5 Hypothetical protein  6 FKBP-typepeptidyl-prolyl cis-trans isomerase (trigger factor)  7 Isocitratedehydrogenase kinase/phosphatase  8 Phage-like element PBSX protein xkdH 9 High-affinity potassium transporter 10 Sarcosine oxidase, alphasubunit 11 Hypothetical protein 12 Phage-like element PBSX protein xkdK13 Phage-like element PBSX protein xkdM 14 Hypothetical protein 15Hypothetical protein 16 Phage protein 17 Hypothetical protein 18Phage-like element PBSX protein xkdQ 19 Ribosomal protein S4 and relatedproteins 20 Phage-like element PBSX protein xkdS 21 Hypothetical protein22 Phage-like element PBSX protein xkdT 23 Tail fiber 24 Heat shockprotein 90 25 Hypothetical protein 26 Bacteriocin uviB precursor 27N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) 28 Enterotoxin 29Membrane protein 30 No hits 31 Thymidylate synthase (EC 2.1.1.45) 32Heat shock protein (dnaJ-2) 33 Hypothetical protein 34 DNA polymerase I35 DNA polymerase I 36 Putative ATP-dependent DNA helicase 37 ABCtransporter ATP-binding protein 38 Terminase large subunit 39 Terminasesmall subunit 40 CobT protein 41 Putative chromosome segregationprotein, SMC ATPase superfamily 42 Deoxycytidylate deaminase (EC3.5.4.12) 43 aceE; pyruvate dehydrogenase e1 component oxidoreductaseprotein 44 Hypothetical protein 45 Hypothetical protein 46 Genomic DNA,chromosome 3, BAC clone: F1D9 47 SWF/SNF family helicase 48 Asparticacid-rich protein aspolin2 49 CDEP 50 Nucleolin 51 Hypothetical protein52 Putative reductase 53 Hypothetical protein 54 Hypothetical protein 55Hypothetical protein 56 Tail fiber 57 gp56 dCTPase 58 Acetate kinase (EC2.7.2.1) 59 GTP-binding protein SAR1 60 Peptide ABC transporter,ATP-binding protein 61 Cytochrome b (EC 1.10.2.2) 62 Homeobox-leucinezipper protein 63 DNA repair protein recN 64 Transposase 65 DNA repairprotein RadA 66 Exonuclease I 67 Transcriptional regulator 68Hypothetical protein 69 Thymidine kinase (EC 2.7.1.21) 70Tryptophanyl-tRNA synthetase (EC 6.1.1.2) 71 CMP-binding factor 723-isopropylmalate dehydratase large subunit (EC 4.2.1.33) 73 DNA ligase74 Putative penicillin-binding protein 75 Signal transducer andactivator of transcription 1 76 Chemotaxis protein CHED 77 Gramicidin Ssynthetase I (EC 5.1.1.11) CPAS-15  1 Gramicidin S synthetase I (EC5.1.1.11)  2 Chemotaxis protein CHED  3 Signal transducer and activatorof transcription 1  4 Putative penicillin-binding protein  5 DNA ligase 6 3-isopropylmalate dehydratase large subunit (EC 4.2.1.33)  7CMP-binding factor  8 Tryptophanyl-tRNA synthetase (EC 6.1.1.2)  9Thymidine kinase (EC 2.7.1.21) 10 Hypothetical protein 11 Hypotheticalprotein 12 Transcriptional regulator 13 Exonuclease I 14 DNA repairprotein RadA 15 Transposase 16 ABC transporter ATP-binding protein 17Y56A3A.29a protein 18 DNA polymerase I 19 DNA polymerase I 20Thymidylate synthase (EC 2.1.1.45) 21 Hypothetical protein 22 SWF/SNFfamily helicase 23 Aspartic acid-rich protein aspolin2 24 CDEP 25Hypothetical protein 26 Terminase large subunit 27 Terminase smallsubunit 28 CobT protein 29 Putative chromosome segregation protein, SMCATPase superfamily 30 Deoxycytidylate deaminase (EC 3.5.4.12) 31 aceE;pyruvate dehydrogenase e1 component oxidoreductase protein 32Hypothetical protein 33 Hypothetical protein 34 DNA repair protein recN35 Homeobox-leucine zipper protein 36 Hypothetical protein 37 No hits 38Hypothetical protein 39 N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)40 Bacteriocin uviB precursor 41 Hypothetical protein 42 Heat shockprotein 90 43 Tail fiber 44 PROBABLE SUCCINYL-COA SYNTHETASE BETA CHAINPROTEIN 45 Phage-like element PBSX protein xkdT 46 Hypothetical protein47 Phage-like element PBSX protein xkdS 48 Ribosomal protein S4 andrelated proteins 49 Phage-like element PBSX protein xkdQ 50 Hypotheticalprotein 51 Phage protein 52 Phage protein 53 Hypothetical protein 54Hypothetical protein 55 Phage-like element PBSX protein xkdM 56Phage-like element PBSX protein xkdK 57 Hypothetical protein 58Sarcosine oxidase, alpha subunit 59 High-affinity potassium transporter60 Phage-like element PBSX protein xkdH 61 Putative nodulation protein62 Isocitrate dehydrogenase kinase/phosphatase 63 FKBP-typepeptidyl-prolyl cis-trans isomerase (trigger factor) 64 Hypotheticalprotein 65 Hypothetical protein 66 Ring-infected erythrocyte surfaceantigen 67 Presumed portal vertex protein 68 GTP-binding protein SAR1 69Hypothetical protein CPLV-42  1 Hypothetical protein  2 Ribosomalprotein S4 and related proteins  3 Hypothetical protein  4 F14H3.11protein  5 Myosin heavy chain, cardiac muscle beta isoform  6 Holin  7N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)  8 Developmentallyregulated GTP-binding protein 1  9 Hypothetical protein 10 DNAinternalization-related competence protein ComEC/Rec2 11 Phage-relatedprotein 12 Antirepressor 13 Phage protein 14 Hypothetical protein 15Excinuclease ABC subunit B 16 Putative oxidoreductase protein 17 DNAtopoisomerase I (EC 5.99.1.2) 18 Imidazole glycerol phosphate synthasesubunit hisF (EC 4.1.3.—) 19 Methyl-accepting chemotaxis protein 20Hypothetical protein 21 Aminomethyltransferase (EC 2.1.2.10) 22Hypothetical protein 23 Hypothetical protein 24 Hypothetical protein 25Hypothetical protein 26 Hypothetical protein 27 Hypothetical protein 28DNA polymerase III, subunit beta 29 Terminase large subunit 30 lin258531 Portal protein 32 Genomic DNA, chromosome 3, BAC clone: F1D9 33Deoxyguanosinetriphosphate triphosphohydrolase (dgtP) 34Virulence-associated protein E 35 Hypothetical protein 363′-phosphoadenosine 5′-phosphosulfate sulfotransferase (PAPSreductase)/FAD synthetase and related enzymes, COG0175 37Phosphoadenosine phosphosulfate reductase (EC 1.8.4.8) 38 Hypotheticalprotein 39 Hypothetical protein 40 WRKY transcription factor 22 41D-threonine dehydrogenase 42 Hypothetical protein 43 Hypotheticalprotein 44 Transcriptional regulator 45 Single-strand binding protein 46Hypothetical protein 47 Sensor histidine kinase CPAS-12  1 Presumedportal vertex protein  2 Ring-infected erythrocyte surface antigen  3Hypothetical protein  4 Hypothetical protein  5 FKBP-typepeptidyl-prolyl cis-trans isomerase (trigger factor)  6 Isocitratedehydrogenase kinase/phosphatase  7 Phage-like element PBSX protein xkdH 8 High-affinity potassium transporter  9 Sarcosine oxidase, alphasubunit 10 Hypothetical protein 11 Phage-like element PBSX protein xkdK12 Phage-like element PBSX protein xkdM 13 Hypothetical protein 14Hypothetical protein 15 Phage protein 16 Hypothetical protein 17Phage-like element PBSX protein xkdQ 18 Ribosomal protein S4 and relatedproteins 19 Phage-like element PBSX protein xkdS 20 Hypothetical protein21 Phage-like element PBSX protein xkdT 22 Tail fiber 23 Heat shockprotein 90 24 Hypothetical protein 25 Bacteriocin uviB precursor 26N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) 27 ABC transporter,permease protein 28 No hits 29 Gramicidin S synthetase I (EC 5.1.1.11)30 Chemotaxis protein CHED 31 Signal transducer and activator oftranscription 1 32 Putative penicillin-binding protein 33 DNA ligase 343-isopropylmalate dehydratase large subunit (EC 4.2.1.33) 35 CMP-bindingfactor 36 Tryptophanyl-tRNA synthetase (EC 6.1.1.2) 37 Thymidine kinase(EC 2.7.1.21) 38 Nucleolin 39 Transcriptional regulator 40 DNA repairprotein RadA 41 Transposase 42 Hypothetical protein 43 Thymidylatesynthase (EC 2.1.1.45) 44 Heat shock protein (dnaJ-2) 45 DNA polymeraseI 46 Putative ATP-dependent DNA helicase 47 ABC transporter ATP-bindingprotein 48 Terminase large subunit 49 Terminase small subunit 50 CobTprotein 51 Putative chromosome segregation protein, SMC ATPasesuperfamily 52 Deoxycytidylate deaminase (EC 3.5.4.12) 53 aceE; pyruvatedehydrogenase e1 component oxidoreductase protein 54 Hypotheticalprotein 55 Hypothetical protein 56 Genomic DNA, chromosome 3, BAC clone:F1D9 57 SWF/SNF family helicase 58 Aspartic acid-rich protein aspolin259 CDEP 60 Nucleolin 61 Alpha/beta hydrolase fold:Esterase/lipase/thioesterase family . . . 62 Normocyte-binding protein 163 Hypothetical protein 64 DNA repair protein recN 65 Homeobox-leucinezipper protein 66 Hypothetical protein 67 gp56 dCTPase

1. A purified bacteriophage preparation comprising four or more C.perfringens-specific bacteriophage, wherein each bacteriophage has lyticactivity against at least 5 strains of a Clostridium species.
 2. Thepurified bacteriophage preparation of claim 1 wherein the Clostridiumspecies is Clostridium septicum.
 3. The purified bacteriophagepreparation of claim 1 wherein the Clostridium species is Clostridiumdifficile.
 4. A purified bacteriophage preparation comprising four ormore C. perfringens-specific bacteriophage, wherein each bacteriophagehas lytic activity against at least 5 strains of a Clostridium specieswhich is antibiotic resistant.
 5. A composition comprising purifiedbacteriophage preparation comprising four or more C.perfringens-specific bacteriophage, wherein each bacteriophage has lyticactivity against at least 5 strains of a Clostridium species and anantibiotic.
 6. The composition of claim 5 wherein the antibiotic isbacitracin
 7. The composition of claim 6 wherein the antibiotic ischlortetracycline.
 8. A method of reducing chicken mortality due to aClostridium infection comprising: administering a purified bacteriophagepreparation comprising four or more C. perfringens-specificbacteriophage, wherein each bacteriophage has lytic activity against atleast 5 strains of a Clostridium species to a chicken.
 9. The method ofclaim 8 wherein the Clostridium species is Clostridium septicum.
 10. Themethod of claim 8 wherein the Clostridium species is Clostridiumdifficile.
 11. The method of claim 8 wherein the method furthercomprises administration of an antibiotic to the chicken.
 12. The methodclaim 11 wherein the antibiotic is bacitracin.
 13. The method of claim11 wherein the antibiotic is chlortetracycline.
 14. The method of claim11 wherein the antibiotic is administered prior to the administration ofthe purified bacteriophage preparation.
 15. The method of claim 11wherein the antibiotic is administered with the administration of thepurified bacteriophage preparation and wherein the antibiotic and thepurified bacteriophage preparation are mixed together.
 16. The method ofclaim 11 wherein the antibiotic is administered after the administrationof the purified bacteriophage preparation.